Ellagic acid and cilostazol ameliorate amikacin-induced nephrotoxicity in rats by downregulating oxidative stress, inflammation, and apoptosis

Amikacin (AK) has the largest spectrum of aminoglycosides. However, its use is constrained because of nephrotoxicity and ototoxicity. Ellagic acid (EA) is a polyphenol present in plants. It has antioxidant, anticarcinogenic, and antimutagenic characteristics. Cilostazol (CTZ) is a phosphodiesterase Ш inhibitor, it is a potent vasodilator and antiplatelet drug. CTZ has an inhibitory effect on reactive oxygen species and superoxide generation in addition to hydroxyl radicals scavenging action. This study determines whether EA and cilostazol have a protective effect against AK-induced nephrotoxicity. Forty-nine rats were divided into seven equal groups: control normal; AK 400 mg/kg; EA 10 mg/kg; CTZ 10 mg/kg; AK 400 mg/kg plus EA 10 mg/kg; AK 400 mg/kg plus CTZ 10 mg/kg; AK 400 mg/kg plus EA 10 mg/kg and CTZ 10 mg/kg. For seven days, drugs were administered using gavage one hour before intramuscular injection of AK. Twenty-four hours after the last AK dosage, blood samples were collected to determine blood urea nitrogen and creatinine levels. Kidneys were removed for histopathological examination and measurement of: malondialdehyde (MDA), catalase (CAT), decreased glutathione (GSH), superoxide dismutase (SOD), interleukin 6 (IL6), tumor necrosis factor-alpha (TNFα), nuclear factor kappa B (NFκB), and Bcl-2 associated x protein (BAX). AK caused kidney damage, inflammatory mediator elevation, and oxidative stress and apoptotic markers. Rats receiving EA or CTZ indicated significant improvement in kidney function, decrease in oxidative stress and inflammation through NF-kB down-regulation and BAX expression. The combination of EA and CTZ showed a synergistic effect. In conclusion, EA and CTZ might play a beneficial role in preventing nephrotoxicity induced by AK partially by inhibition of tissue inflammation and apoptosis.


Introduction
Nephrotoxicity is defined as a 50% increase in serum creatinine or a 50% decrease in creatinine clearance and an increase in blood urea nitrogen. Drugs like aminoglycosides, chemotherapeutic a1111111111 a1111111111 a1111111111 a1111111111 a1111111111

Collection of blood and renal samples
On the 8 th day of the experiment, the animals were anesthetized using a 50 mg/kg intraperitoneal injection of sodium pentobarbital for sacrifaction. The blood samples from the retro-orbital plexus of veins were collected using microcapillary tubes. These samples were centrifuged at 3000 × g for ten minutes to separate the serum for determining serum BUN and creatinine concentrations. The left kidneys were dissected for histopathological examination and biochemical estimation of malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), nuclear factor kappa B (NFκB), interleukin 6 (IL6), tumor necrosis factor-alpha (TNFα), and Bcl-2 associated x protein (BAX). The remaining kidneys were frozen at −80˚C and ice-cold 0.05 M phosphate buffer pH 7.4 until used.

Histopathological studies
The kidneys were quickly extracted and opened. The specimens were fixed in 10% formalin, sectioned into 5 mm thick paraffin blocks, and hematoxylin as well as eosin stains (H&E) were used for light microscopy [20].

Statistical analysis
To compare all groups, a one-way analysis of variance (ANOVA) was conducted, while to compare between every two groups, the post-hoc Turkeyʼs test was used. All data are expressed as mean ± SEM. A p-value less than 0.05 is considered significant. Computer analysis of the obtained data was conducted using the Statistical Package for Social Services version 25 (SPSS).

Effect of ellagic acid, cilostazol and their combination on renal function
AK 400 mg/kg significantly increased BUN and creatinine levels compared with the normal control group. EA 10 mg/kg or CTZ 10 mg/kg alone produced a non-significant reduction in both parameters in relation to the normal control group. AK plus EA and AK plus CTZ significantly reduced BUN and creatinine compared with the AK group. AK plus EA and CTZ significantly reduced both parameters than each drug alone (Fig 2).

Effect of ellagic acid, cilostazol, and their combination on oxidative stress markers
AK 400 mg/kg significantly increased MDA levels in the renal tissue and caused a significant reduction of CAT, SOD, and GSH in renal tissue compared to the control group. EA10 mg/kg or CTZ 10 mg/kg alone produced non-significant results concerning the normal control . Ellagic acid (EA) 10 mg/kg, cilostazol (CTZ) 10 mg/kg and their combination were administered one hour before intramuscular injection of amikacin 400 mg/kg for seven days. Groups were compared using one-way ANOVA and post-hoc Turkeyʼs test. Values are presented as mean ± SE (n = 7). Values without common small letters are significantly different (p < 0.05).
https://doi.org/10.1371/journal.pone.0271591.g002 group. AK plus EA and AK plus CTZ significantly decreased MDA and significantly increased GSH, SOD, and CAT in renal tissue concerning the AK group. AK plus EA and CTZ produced a more significant MDA reduction and a more significant rise of GSH, SOD, and CAT than each drug alone (Fig 3).

Effect of ellagic acid, cilostazol, and their combinations on inflammatory markers
AK 400-mg/kg produced a significant increase in renal tissue TNFα and IL6 compared to the normal control group. EA 10 mg/kg or CTZ 10 mg/kg alone produced non-significant results concerning the control group. AK plus EA and AK plus CTZ produced a significant reduction in TNFα and IL6 in renal tissue compared to the AK group. AK plus EA and CTZ produced a more significant decrease in TNFα and IL6 than each drug alone (Fig 4).

Effect of ellagic acid, cilostazol, and their combination on apoptotic markers
AK 400 mg/kg resulted in a significant increase in NFκB and BAX expression in renal tissue compared with the normal control group. EA 10 mg/kg or CTZ 10 mg/kg alone produced a non-significant result concerning the normal control group, while AK plus EA and AK plus CTZ showed a significant decrease in NFκB and BAX expression concerning the AK group. AK plus EA and CTZ produced a more significant NFκB reduction and BAX expression in renal tissue than each drug alone (Fig 5).   EA 10 mg/kg produced no changes in normal kidney structure [C]. Also, CTZ 10 mg/kg produced no changes in normal kidney structure [D]. The administration of EA 10-mg/kg or CTZ 10 mg/kg one hour before AK as a prophylactic agent exhibited an improvement of AKinduced nephrotoxicity in reducing the percentage of the area of inflammation, eosinophilia, and necrosis [E]&[F] respectively. Administration EA plus CTZ one hour before AK as a prophylactic agent showed more reduction in the percentage of the area of inflammation, eosinophilia, and necrosis [G] than each drug alone (Fig 6).

Discussion
AKI occurs in 20-30% of children exposed to aminoglycosides [21]. This study investigated the renoprotective effect of EA alone, CTZ alone, and their combination.
The results of the current work showed that AK 400 mg/kg significantly increased serum BUN and creatinine levels. These results agree with Hlail and colleagues [1], who demonstrated than intraperitoneal AK 120 mg/kg injection for 14 d produced a significant increase in serum creatinine and urea levels. Also, previous studies found that i.m injection of AK 100 mg/kg for seven days produced a significant rise in creatinine, uric acid, and urea [5].
Multiple pathophysiological effects of AK-induced kidney damage include the creation of reactive oxygen and nitrogen species and stimulation of apoptosis, as AK forms a complex with mitochondrial Fe2+, causing the development of free radicals. These free radicals and reactive species are essential in drug-induced renal impairment and BUN and creatinine increase [7].
Antioxidant enzymes like SOD and CAT are essential for cellular antioxidative defense. SOD catalyzes the formation of hydrogen peroxide (H 2 O 2 ) by superoxide radical dismutation [22]. MDA is a lipid peroxidation end product that can be used as a biological biomarker to describe the degree of oxidative stress [23]. GSH acts as a potent electron donor acting against free radicals. With the aid of glutathione peroxidase enzymes, GSH can degrade H 2 O 2 to H 2 O [24].
Also, this work indicated that AK 400 mg/kg produced a significant reduction in the antioxidant parameters; GSH, SOD, and CAT, and a significant increase in oxidation parameter; MDA, in renal tissue. These results are following Abdel-Daim and colleagues [5] who reported a significant elevation of MDA and a significant reduction of SOD, CAT, and GSH caused by AK 100 mg/kg. AK is not metabolized in the body and is primarily eliminated in the urine. As a result, it accumulates in proximal tubules and glomeruli, leading to the activation of renin-angiotensinaldosterone system, lowering the glomerular filtration rate and increasing the production of platelet-activating factor, reactive oxygen species (ROS), and vasoconstrictors [25]. The excessive ROS production causes oxidative stress, which causes significant interconnected disturbances in cellular metabolism, such as protein and nucleic acid structure changes, DNA damage, apoptosis induction, elevation in intracellular free calcium, damage to membrane ion transport, and cell damage from lipid peroxidation [1].
TNF is a proinflammatory cytokine formed by macrophages and monocytes and can activate neutrophils and lymphocytes, enhancing vascular endothelial cell permeability, and triggering the production and release of other cytokines. It acts on tumor necrosis factor receptor 1 (TNFR1) and 2 (TNFR2). TNFR1 mediate inflammation and increases fibroblast proliferation by activating nuclear factor (NF). TNFR2 contributes to cell migration, regeneration, proliferation, and TNF1-mediated apoptosis regulation. TNFα may stimulate the NF-B pathway, which regulates the transcription and production of inflammatory mediators. This is a vicious cycle that exacerbates inflammatory reactions [26].
The results of this work proved that AK 400 mg/kg produced a significant increase in NFκB, TNFα, and IL6 in relation to the normal control group. Ozbek et al. (2009) agreed with these results and stated that intraperitoneal injection of gentamycin 100 mg/kg significantly increased NFκB expression in renal tissue [27].
AK-induced nephrotoxicity could be due to up-regulation of TNF-α expression or due to AK-induced oxidative stress, which induces oxygen-containing derivatives and cytokine production, which function as a second messenger for activating NF-B, resulting in the transcription of cytokines, growth factors, and extracellular matrix proteins [5] This work indicated that AK 400 mg/kg significantly increased BAX expression in renal tissue. Helmy et al. (2020) agreed with this result and showed that AK 1.2 g/kg single intraperitoneal injection increased BAX expression in renal tissue [28].
Aminoglycosides can cause apoptosis in the kidney by increasing the content of cytosolic BAX protein, which activates the mitochondrial pathway of apoptosis, it includes caspase-9 activation as an initiator, caspase-3 activation as an effector, and DNase activation, leading to DNA fragmentation and apoptosis [29].
In this study, oral administration of EA 10 mg/kg one hour before AK significantly reduced BUN and creatinine. These findings support those of [14], who reported that EA 10 mg/kg significantly reduced urea and creatinine in gentamycin 100 mg/kg induced nephrotoxicity. Ateşşahín et al. (2007) [30] reported that EA 10 mg/kg significantly reduced urea and creatinine in nephrotoxicity induced by intraperitoneal injection of cisplatin 7 mg/kg. The improvement in RBF and GFR could explain EA's favorable effect in improving kidney function tests and lowering creatinine and BUN levels [31].
In this study, EA 10 mg/kg orally one hour before AK produced a significant reduction of oxidation parameter; MDA, and a significant increase in the antioxidant parameters; GSH, CAT, and SOD in renal tissue in relation to the AK group. A previous study [14] agreed with these results as they reported that EA 10 mg/kg induced a preventive effect on nephrotoxicity caused by gentamycin as it increased SOD, CAT, and GSH levels. Also, Bhattacharjee et al. (2021) reported that oral administration of EA 25, 50 mg/kg, orally for two months showed a preventive effect on nephrotoxicity caused by lead by increasing CAT, SOD, GSH, and reducing MDA compared with the control nephrotoxic group [32].
The ability of EA to scavenge free radicals has been related to its intrinsic antioxidant activity. This is since it can transfer the phenolic H-atom to a free radical. Lactone systems and EA hydroxyl groups can create hydrogen bonds and act as hydrogen donors and electron acceptors. As a result, EA can participate in antioxidant redox reactions, resulting in a highly efficient free radical scavenger [33].
Oxidative stress has been shown to be decreased by EA through modulation of several mechanisms. These involve antioxidant response activation through Nrf2, suppression of cytokines, such as IL1, IL6, TNF, and cyclooxygenase 2 (COX-2) through NF-kB, and cell survival or apoptosis control through NF-kB [34]. EA is classed as a multiple-function antioxidant since it exerts its beneficial effect through both primary and secondary ways [35].
EA 10 mg/kg orally one hour before AK significantly reduced TNFα, IL6, and NFκB expression in this work. These findings are consistent with that of Marn et al. (2013), who suggested that EA reduced NF-B, IL-6, and TNF levels compared to the control group in mice with ulcerative colitis [36]. EA inhibits inflammation through modulating the NF-B signaling pathway [37]. These findings are consistent with Cornélio Favarin et al. (2013), who discovered that EA 10 mg/kg increased the anti-inflammatory cytokine IL-10 and decreased the proinflammatory cytokine IL-6 in bronchoalveolar lavage fluid [38]. EA decreases toll-like receptor 4 (TLR4) and high mobility group protein 1 (HMGB1) in the kidney tissue by cutting down TLR4 downstream protein leading to reduction in inflammatory factors [26].
In this study, EA 10 mg/kg orally one hour before AK reduced BAX expression in renal tissue. A previous study [14] agreed with this finding and reported that EA 10 mg/kg reduced gentamycin-induced nephrotoxicity in rats by increasing Bcl2/BAX ratio and decreasing Caspase-3. It is one of the main executors of apoptosis.
EA's antioxidant and anti-apoptotic qualities may be attributed to the increased SIRT1 expression in renal tissues [39]. SIRT1 (sirtuin1) is the mammalian homolog of the yeast Sir2 (silent information regulator 2). It protects against oxidative stress by deacetylating forkhead box O (FOXO) and tumor suppressor protein (p53). SIRT1 deacetylates p53 and FOXO, resulting in transcriptional activities suppression and loss of stress-induced apoptosis [40]. FOXOs also contribute to the viability of cells through the transactivation of enzymes that detoxify ROS, such as SOD2/MnSOD and CAT [39].
Also, this study indicated that oral administration of CTZ 10 mg/kg one hour before AK significantly decreased BUN and creatinine. These findings support a previous study [15] which demonstrated that administration of CTZ 10 mg/kg once daily for eight days reduced creatinine, urea, and uric acid levels in the nephrotoxicity induced by gentamycin. Also, Gokce et al. (2012) reported concomitant use of CTZ 10 mg/kg, orally with cyclosporine reduced urea and creatinine level [41].
This work showed that oral administration of CTZ 10 mg/kg one hour before AK produced a significant decrease in oxidation parameter; MDA and a significant increase in the antioxidant parameters; GSH, CAT, and SOD in renal tissue in relation to the AK group. These results agree with that of Gokce et al. (2012), who reported that administration of CTZ 10 mg/ kg for seven days eases cyclosporine-induced nephrotoxicity by decreasing MDA and increasing SOD and CAT activity [41]. CTZ prevents oxidative stress by activating redox defense systems through increased expression of PI3K/Akt and Nrf2/HO-1 mRNAs, resulting in oxidative stress reduction and restoration of mitochondrial dysfunction [12].
In this study, CTZ 10 mg/kg orally as a prophylactic dose, significantly reduced expression of BAX in relation to AK group. These results agree with a previous study [15] which reported that CTZ 10 mg/kg for eight days produced a significant reduction in BAX expression in gentamycin-induced nephrotoxicity model. CTZ suppresses signals of mitochondria-dependent apoptosis. Additionally, it reduces cytochrome c release from mitochondria and down-regulates BAX expression [45].
This histopathology findings revealed that AK 400 mg/kg was associated with disturbances in the kidney histopathological picture, including inflammatory cell infiltration, tubular epithelial lining degeneration, and tubular necrosis. These results agree with Abdel Fattah and Gaballah, (2020), who demonstrated that marked degenerative changes in the kidney and marked tubular necrosis occurred with AK [46]. EA administration before AK exhibited an improvement in the histopathological changes as it decreased inflammation and necrosis. These results are consistent with Bhattacharjee et al., 2021, who stated that EA 25, 50 mg/kg reduced histopathological changes and renal tubular necrosis in lead-induced nephrotoxicity [32]. Also, CTZ administration one hour before AK shows a reduction in inflammation and tubular necrosis. These results agree with Abdelsameea and colleagues [15] who reported that administration of CTZ 10 mg/kg rat eases degenerative changes in the renal cortex in gentamycin-induced nephrotoxicity model.
In this study, oral administration of EA 10 mg/kg plus CTZ 10 mg/kg before AK 400 mg/kg produced a more significant reduction of BUN and creatinine; more significant reduction of oxidation parameter MDA; more significant reduction of antioxidant parameters: GSH, SOD, and CAT; more significant reduction of inflammatory mediators: TNFα, IL6; more significant reduction of NFκB and BAX expression; more improvement in the histopathological changes developed in kidney tissue by AK than each drug alone due to the synergistic effect of both drugs.

Conclusion
EA and CTZ have a renoprotective effect partially due to their antioxidant, anti-inflammatory, and anti-apoptotic effects.